Human stratified squamous epithelia differ in cellular fatty acid composition

Journal of Dermatological Science 24 (224) 14 – 24 www.elsevier.com/locate/jdermsci

Human stratified squamous epithelia differ in cellular fatty acid composition Hiroto Terashi, Kenji Izumi, Lenore M. Rhodes, Cynthia L. Marcelo * Di6ision of Plastic and Reconstructi6e Surgery, Department of Surgery, Uni6ersity of Michigan Medical Center, Ann Arbor, MI, USA Received 2 June 1999; received in revised form 20 December 1999; accepted 27 December 1999

Abstract The phospholipid component of the cellular membrane is crucial to the structure and function of cells. Basal cells from three epithelial tissues, adult human skin epidermis, oral mucosa, and hair follicles, grow rapidly in serum- and lipid-free medium. Analysis of phospholipid extracts from the above three types of stratified squamous epithelium in both in vivo and in vitro was done to relate fatty acid cell composition to cell function. The fatty acid composition of hair follicles in vivo was analyzed in plucked scalp hairs, and those of skin epidermis and oral mucosa in vivo were analyzed after separating the tissue into suprabasal and basal layers. The fatty acid composition of the in vivo cells from hair follicles shows a partial essential fatty acid (EFA)-deficient state. There was no significant difference between the skin epidermis and the oral mucosa in the fatty acid composition of the in vivo cells from each basal layer. However, in the suprabasal layers, the percent of linoleic acid (18:2) from the skin epidermis was higher than that from the oral mucosa. This study shows that total fatty acid composition in cell membranes of stratified squamous epithelium varies with their keratinization pattern. When cultured, the three types of rapidly growing keratinocytes showed the same essential fatty acid deficient pattern in the membrane phospholipids. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Phospholipids; Epidermal keratinocytes; Oral mucosal cells; Hair follicle cells; Linoleic acid (18:2); Cell membrane

1. Introduction Normally, human stratified squamous epithelium undergoes three types of keratinization;

orthokeratinization in skin epidermis, parakeratinization in oral mucosa, and trichilemmal keratinization in hair follicles. The morphologic and biochemical differences between

Abbre6iations: BPE, bovine pituitary extracts; DMEM, Dulbecco’s modified Eagles medium; EFA, essential fatty acid; EGF, epidermal growth factor; FAMEs, fatty acid methyl esters; FBS, fetal bovine serum; GC, gas chromatography; P, passage; PBS, phosphate-buffered saline; PUFA, polyunsaturated fatty acids; TLC, thin-layer chromatography; 15HETE, 15-hydroxy5,8,11,13,eicosatetraenoic acid; 16:0, palmitic acid; 18:1(n-7), oleic acid; 18:2(n-6), linoleic acid; 20:4(n-6), arachidonic acid. * Corresponding author. Present address: University of Michigan Medical School, 5659 Kresge I, Ann Arbor, MI 48109-0592, USA. Tel.: + 1-734-7633473; fax: + 1-734-6479666. 0923-1811/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 9 2 3 - 1 8 1 1 ( 0 0 ) 0 0 0 7 7 - 3

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epidermis and oral mucosa or hair follicles have been studied: the variations in histology [1], in keratin pattern [2–6], and in protein expression [7] have been reported. However, the lipid content of the cells forming these three different tissues has not been extensively investigated. One family of lipids, the fatty acids, is incorporated into the phospholipids that form the cell membranes, including the plasma membrane and the mono-di- and triglyceride compartments with the cell. These long chained lipids are the precursors for the eicosanoid family of inflammatory agents, and are involved in the generation of one group of second messengers, the protein kinase C system [8]. The human body can synthesize the saturated fatty acids, such as palmitic acid (16:0) and stearic acid (18:0) as well as other fatty acids up to the chain length of C26 [9] from glucose and acetate-carbon sources [10]. Monounsaturated fatty acids, mainly palmitoleic acid (16:1) and oleic acid (18:1), are also produced from precursor saturated fatty acids by the usual desaturation enzymes [9]. However, the major essential fatty acids, linoleic acid (18:2) and arachidonic acid (20:4), cannot be synthesized [11]. These must be supplied by the diet because the mammalian system is incapable of inserting double bonds beyond the n-9 position [9]. Arachidonic acid (20:4) is essential as a source of leukotrienes [12] and prostaglandins [13]. Interestingly, it is also reported that 20:4 is actively converted from 18:2 within the cultured epidermal keratinocytes in the extreme dietary state of essential fatty acid (EFA)-deficient medium [10,14]. When incorporated into the C1 and C2 carbons of phospholipids, fatty acids help determine the structure and function of cell membranes. Our hypothesis is that the length and saturation state of the fatty acids forming the membranes, especially the quantity and type of essential fatty acid, can regulate cell growth and differentiation as well as the function of cells. We have reported that adult human epidermal cells in culture are EFA-deficient when grown using serum-free medium, and that normalization of the cultured epidermal keratinocytes by adding essential, polyunsaturated fatty acids (PUFAs) to

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serum-free medium alters the growth and differentiation of the cultures [15,16]. Hair follicle cells are not only a progenitor of interfollicular epidermal cells [7,17,18], but are also the cells at the bulge area in the outer root sheath which are regarded as skin pluripotent stem cells. These cells are the source of hair itself and sebaceous gland [19–21]. In addition, it is unclear why the outer root sheath cells in hair follicles proliferate without epidermal differentiation as they surround the hair shaft. We theorized that the fatty acid esterified into the phospholipids forming the cell membranes could be involved in these specialized cell functions. Our purpose in this study was to compare the fatty acid composition of membrane phospholipids in the in vivo skin epidermis to that of hair follicles. We also wanted to determine the effect of growth of these two epithelia in EFAdeficient medium on the levels of these membrane lipids. We also studied oral mucosal cells, which are another type of keratinocytes with specialized functions and keratinization pattern [6].

2. Materials and methods

2.1. Materials Heptadecanoic acid (17:0), methylated fatty acid standards, and 16:0, 18:1(n-7), 18:2(n-6), and 20:4(n-6) were obtained from Nu-CHEKPREP, Elysian, MN. Amino acids and medium reagents for MCDB 153, fetal bovine serum (FBS), trypsin type IX, trypsin inhibitor type I-S, gentamicin (10 mg/ml), and bovine collagen type I, were obtained from Sigma, St Louis, MO. Plastics (T-75 flask) were from Laboratory Science, Corning, NY. Petroleum ether (30–60°C), and benzene were purchased from Baker, Phillipsburg, NJ. Methanol and chloroform were obtained from B&J Baxter, McGraw Park, IL. Thin-layer silica gel 60 plates were obtained from Merck, Darmstadt, Germany. Frozen bovine pituitaries were from Pel-Freeze, Rogers, AR, and filters were obtained from Millipore, Bedford, MA.

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2.2. Human stratified squamous epithelium

2.3. Primary cell culture

The tissues studied were adult human epidermis, oral mucosa, and hair follicles. Human epidermis was obtained from fresh surgical resection specimens from face-lift operations and oral mucosa was the remnant of gingival tissue from the extraction of teeth for orthodontic reasons. The deep dermis and the subcutaneous tissue of skin samples were used for culturing hair follicle cells. To obtain skin epidermal cells and oral mucosa epithelial cells, the skin and mucosal samples were cut into small strips without deep dermis and subcutaneous tissue, and then digested overnight at room temperature with 0.03% trypsin. The tissue was then separated at the epidermal-dermal junction, leaving the basal cells on the dermal layer [22,23]. The suprabasal cell component was separated, and frozen in methanol for lipid extraction. The basal cells were then gently scraped from the dermis into MCDB 153 medium plus 10% FBS. The cell suspension was centrifuged. The pellet was either used to grow primary cell cultures (see next section) or was fixed in methanol and frozen at −80°C until lipid extraction. The in vivo hair follicles (plucked hair) consisted of 30–40 plucked scalp hairs from each of four volunteers. Each individual hair was divided into two fractions as follows (Fig. 1): one was the portion of the hair shaft above the skin surface (section 1); the second was the entire hair unit below the skin surface, but not including hair bulb area (section 2). Each fraction was ground while frozen under liquid nitrogen and homogenized in methanol.

Bovine pituitary extract (BPE) is a 0.15-M NaCl homogenate of frozen powdered pituitaries that was clarified by ultracentrifugation, twice, at 200 000× g, and filtered through 0.8-, 0.45-, and sterile 0.22-mm filters [15]. The basic medium, MCDB 153, was prepared as described by Boyce and Ham [24] and was supplemented with 0.6× 10 − 6 M (0.218 mg/ml) hydrocortisone, 5 ng/ml epidermal growth factor (EGF), 5 mg/ml insulin, 6 mg% BPE, and 0.15 mM CaCl2 to form MCDB 153 complete medium. The cell pellets for culturing adult human epidermis and oral mucosa were prepared as described in the previous section and were grown as previously by us [15,25]. The method for culturing hair follicle cells was modified from the techniques described by Rochat et al. [18]. The hair follicles were isolated from the deep dermal fragments under a dissecting microscope and were carefully transferred onto bovine type I collagen coated plastic dish with Dulbecco’s modified Eagles medium (DMEM) plus 10% FBS. On day 4, the culture medium was changed to MCDB 153 complete medium without serum as previously described by us [26].

2.4. Subculti6ation The cell cultures were fed every 48 h until almost 70% confluence was reached. The cells were passaged by incubation at 37°C with 0.03% trypsin-0.01% EDTA and plated at 3–4× 106 cells per T-75 flask. The keratinocytes from epidermis were passaged to P1 7 days after primary culture. The keratinocytes from hair follicles were passaged to P1 after 14 days of primary culture. Samples of the primary, P0 oral keratinocytes were not prepared for lipid analysis because the amount of material was limited and only P1–3 cultures were analyzed.

2.5. Lipid analysis Fig. 1. Human scalp hair. Section 1 shows the portion of the hair shaft above the skin surface, and section 2 shows the entire hair unit below the skin surface, but not including hair bulb area.

Primary and passage 1–3 keratinocytes were rinsed twice with calcium-free, phosphatebuffered saline (PBS) and were scraped into methanol. The preparations from tissues (in vivo)

H. Terashi et al. / Journal of Dermatological Science 24 (2000) 14–24

and in vitro grown cells were extracted with a 1:2:1.5 ratio of methanol:chloroform: 0.1 M KCl in 50% methanol, and the organic phase was re-extracted with 2.5× volume of 0.1 M KCl in 50% methanol [15]. Each precipitated protein was measured by modified Lowry protein assay [27]. The extracted fraction was suspended in 75 ml of 1:1 chloroform:methanol after evaporating under a nitrogen stream, applied to a thin-layer chromatography (TLC) plate, and chromatographed in one direction using chloroform:methanol:glacial acetic acid (90:8:1). In our previous studies, we have found that the fatty acid composition of the cellular phospholipid fraction reflects the percent fatty acid amounts of all the fatty acid-containing lipids in the cells [15]. We compared the fatty acid percentage composition in the total lipid extracted from the tissues and cells in culture to a paired TLC extracted fraction. In our analysis protocol, acetic acid in our TLC procedure did not change the % PUFAs when compared to the original extracted lipid fraction. When analyzing the hair follicle cell samples, it was important to extract the cell phospholipids because the tissue material is covered with a complex, long-chained lipid coat secreted by associated sebaceous glands. Therefore, all our lipid analyses were done on the TLC-separated phospholipid fraction. After TLC chromatography of the cell lipids, the phospholipid containing area of the plate was scraped and material was eluted from the silica during transmethylation with 6% methanolic-HCl. A total of 50 mg 17:0 was added at this time (internal standard) and the sample heated for 3.5 h at 80°C in order to form fatty acid methyl esters (FAMEs). The FAMEs were resuspended in 200 ml of benzene, filtered using a 0.45-mm filter, evaporated, and resuspended in 50 – 150 ml (as determined by the quantity of protein) filtered chloroform for analysis. A total of 0.5 ml of chloroform was injected for analysis.

2.6. Analysis of FAMEs FAMEs were analyzed using a Shimazu gas chromatograph (GC) model GC-14A equipped with a J and W Scientific (Folsom, CA) fused

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silica Megabore DB225, 0.53-mm diameter column. The FAMEs were eluted with scrubbed helium at a flow rate of 2.79 ml/min at 210°C for 16 min, at a gradient of 4°C/min until 220°C, and then isothermic until 18.5 min. The flame ionization detector output of the gas chromatograph was digitized by an IBM-PC computer interface (model AN-146, Alpha Products, Darien, CT). Both the recording and data evaluation software were written by us in BASIC or FORTRAN.

3. Results

3.1. The fatty acid composition of skin epidermal keratinocytes In Table 1, the fatty acid composition of epidermal cells isolated from skin (the suprabasal and basal pellets) and in vitro first passage (P0) and passage P1–3 keratinocytes is compared. The data are expressed as mg of a fatty acid per mg of protein (data not shown). The values were normalized as percent of the total fatty acid in the sample (mg fatty acid/total fatty acid present). This normalization is done so that tissue variations in the fatty acid amount extracted per mg protein (or DNA or cell) were negated and a direct comparison among stratified tissues, non-stratified tissues and cells in culture could be done. A total of 19 biologically occurring fatty acids plus the 17:0 standard were identified and quantified by gas chromatography. The basal cell pellet values were compared to the suprabasal cell pellet and to the P0 (3 and 7 days after primary platting) and to P1–P3 values using a two-tailed Student’s t-test. The comparison was to the basal pellet because it is the source of the cell type that grows in culture. The P1–P3 values were combined because we observed, as previously reported, that the keratinocytes became essential fatty acid (EFA)-deficient by the first passage [15], and that any difference in fatty acid amounts between P0 and subsequent passages was not significant (data not shown). The ratio of PUFAs (18:2, 20:2, 20:3, and 20:4) to the monounsaturated fatty acids (14:1, 16:1, 18:1, 20:1, and 24:1) was 33:19

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Table 1 Fatty acid content of epidermal keratinocytes Fatty acid

Percent total lipids In vivo pellet

14:0 14:1 16:0 16:1, 18:0 18:1, 18:2, 18:3, 20:0 20:1, 20:2, 20:3, 20:4, 20:3, 22:0 22:1, 22:6, 24:0 24:1, Sat Unsat Mono Poly n

n-7a n-9 n-6 n-6 n-9 n-6 n-6 n-6 n-9 n-9 n-3 n-9

P0

P1–P3

(Suprabasal)

(Basal)

(3 Days)

(7 Days)

0.99 0.1b 0.89 0.2b 17.5 90.3b 0.99 0.2b 16.0 90.2f 16.0 90.8b 27.4 90.5d 0.19 0.1b 1.59 0.0b 0.39 0.0b 0.89 0.2b 2.69 0.2b 6.3 90.3b 0.09 0.0b 2.49 0.0b 0.19 0.0b 1.29 0.1b 4.19 0.1b 0.89 0.0b 42.4 90.4c

1.19 0.1 0.29 0.1 19.19 0.3 0.99 0.0 18.19 0.4 15.79 1.0 20.79 0.9 0.09 0.0 1.7 9 0.1 0.49 0.0 0.69 0.0 3.79 0.3 8.49 0.5 0.09 0.0 2.8 9 0.4 0.39 0.1 1.49 0.0 3.7 9 0.2 1.49 0.1 46.39 0.5

1.6 9 0.1b 0.1 90.0b 20.0 90.4b 2.4 90.3b 15.4 90.2c 19.2 90.2b 19.2 90.7b 0.0 90.0b 1.0 90.0b 0.6 90.0f 1.3 90.0d 3.2 90.3b 7.8 90.1b 0.0 90.0b 1.4 90.1b 0.3 90.1b 1.5 90.1b 3.5 90.2b 1.5 90.0b 43.0 90.4c

1.8 90.1c 0.0 90.0b 19.4 9 0.9b 4.9 90.4c 15.8 9 0.3b 25.0 9 0.8d 13.7 9 0.4d 0.0 90.0b 0.8 90.0c 0.5 90.0b 0.9 9 0.0b 3.0 90.4b 6.3 90.5b 0.0 90.0b 0.9 90.2b 0.5 90.2b 1.4 90.1b 3.5 90.1b 1.5 90.0b 42.2 9 1.4b

3.4 90.1e 0.2 90.1b 23.2 9 0.3d 9.2 90.2e 14.5 9 0.2c 34.1 9 0.4e 2.2 90.1d 0.0 9 0.0b 0.7 90.1c 0.3 9 0.0b 2.2 90.1c 1.5 9 0.1c 3.1 90.1d 0.0 9 0.0b 0.6 90.0b 0.3 90.0b 1.3 9 0.1b 1.3 90.0d 2.0 9 0.1b 43.6 90.3b

57.6 90.4c 18.9 90.8b 37.2 90.5b 4

53.79 0.5 18.9 9 0.8 33.49 0.9 4

57.0 90.4c 24.0 9 0.3c 31.5 90.5b 3

57.8 9 1.4b 32.5 9 1.3d 23.9 9 0.5d 3

56.4 9 0.3b 46.1 9 0.4e 9.0 90.2e 14

a

The (n-) nomenclature indicates the double bond closest to the methyl end. Not significant. c 0.05\P\0.01. d 0.01\P\0.001. e PB0.001. f P $ 0.05. b

in the basal cell pellet, and the ratio shifted gradually down to 9:46 in the P1 – P3 cells. As previous reported [15], the PUFAs were significantly decreased, whereas the monounsaturated ones, mainly 16:1 and 18:1, were increased. The keratinocytes grown in the fatty acid- and serum-free medium became essential fatty acid-deficient, as previously reported for this cell culturing system [15], with the shift to EFA-deficiency beginning at the primary plating, P0. In addition, the percent of linoleic acid (18:2) in the suprabasal cell pellet was significantly greater than that measured in the basal cell pellet.

3.2. The fatty acid composition of oral mucosal keratinocytes A total of 19 fatty acids were identified and quantified in the oral mucosal keratinocyte samples. The basal cell pellet values were compared to the tissue sample and to the suprabasal cell pellet fatty acid percent, and to the P1–3 cell culture values using a two-tailed Student’s t-test (Table 2). There was little significance difference among the fatty acids measured in the suprabasal and the basal cell pellets, and the oral mucosal tissue. The change from in vivo tissue fatty acid levels toward

H. Terashi et al. / Journal of Dermatological Science 24 (2000) 14–24

an EFA-deficient state was as seen in epidermal keratinocyte cultures.

3.3. The fatty acid composition of hair follicle cells The portion of the hair shaft directly above the skin surface (Fig. 1, section 1) had little phospholipid (data not shown), as previously reported [28]. Thus, the phospholipids of the second hair portion taken from below the skin (Fig. 1, section 2) are derived only from the hair follicles, including the layers of the hair follicles [29]. The total

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fatty acid content of the phospholipids extracted from the hair follicle cells is shown in Table 3. The in vivo, plucked hair fatty acid values were compared to the P0 (14 days) and P1–P3 values using a two-tailed Student’s t-test. Only 20:4-fatty acid levels differed between the in vivo plucked hair and the P0 primary samples. Inspection of the data presented in Table 3 shows that the ratio of saturated to unsaturated fatty acid changed from 64:36 in vivo to 44:56 in vitro. The percent of palmitic acid (16:0), one of the saturated fatty acids, is extremely high (44.19 2.2%; mean9 S.E.M.) in the in vivo hair sample

Table 2 Fatty acid content of oral mucosal cells Fatty acid

Percent total lipids In vivo pellet

14:0 14:1 16:0 16:1, 18:0 18:1, 18:2, 18:3, 20:0 20:1, 20:2, 20:3, 20:4, 20:3, 22:0 22:1, 22:6, 24:0 24:1, Sat Unsat Mono Poly n a

n-7a n-9 n-6 n-6 n-9 n-6 n-6 n-6 n-9 n-9 n-3 n-9

P1–P3

(Whole)

(Suprabasal)

(Basal)

1.290.0b 0.09 0.0b 23.09 0.4b 4.39 0.2e 16.99 0.6b 20.39 0.5b 14.59 0.7b 0.0 9 0.0b 0.39 0.1b 0.09 0.0b 0.3 9 0.1b 3.39 0.1d 9.39 0.4b 0.0 9 0.0b 0.79 0.1b 0.5 9 0.1b 1.9 9 0.2b 2.49 0.1b 1.19 0.2b 44.49 0.8b

1.4 90.2b 0.29 0.1b 23.89 0.3b 3.69 0.5b 15.9 90.4b 17.59 0.7b 18.69 0.6b 0.0 90.0b 1.19 0.1b 0.59 0.1b 0.6 90.1b 2.1 90.1b 6.5 90.2b 0.0 90.0b 1.4 90.0c 0.0 90.0b 1.8 90.0b 4.0 90.3b 1.2 90.1b 47.69 0.6b

1.0 9 0.3 0.0 90.0 29.7 9 1.9 2.2 90.4 19.4 9 1.1 16.0 91.6 15.1 90.9 0.1 90.0 0.4 90.1 0.2 90.1 0.2 90.1 0.7 90.3 9.2 91.1 0.0 90.0 0.5 90.1 0.2 9 0.1 1.3 90.2 2.1 90.3 0.9 90.2 53.0 9 2.7

3.0 90.1c 0.1 9 0.0b 18.6 9 0.2e 9.0 90.2d 14.8 9 0.4b 39.9 9 0.7d 2.0 9 0.1d 0.1 90.0b 0.0 9 0.0b 0.5 90.1b 1.9 9 0.2c 2.8 9 0.3c 2.9 90.0e 0.0 9 0.0b 0.0 90.0b 0.8 90.2b 1.4 90.1b 0.9 90.1b 1.3 90.1b 37.3 90.5e

55.69 0.8b 26.39 0.9b 27.39 0.8b 4

52.49 0.6b 22.99 1.0b 27.89 0.9b 5

47.0 9 2.7 19.5 9 1.9 25.3 9 1.5 5

62.7 9 0.5e 51.6 90.8d 9.7 90.4c 5

The (n-) nomenclature indicates the double bond closest to the methyl end. Not significant. c 0.05\P\0.01. d 0.01\P\0.001. e P $0.05 b

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Table 3 Fatty acid content of hair follicle cells Fatty acid

14:0 14:1 16:0 16:1, 18:0 18:1, 18:2, 18:3, 20:0 20:1, 20:2, 20:3, 20:4, 20:3, 22:0, 22:1, 22:6, 24:0 24:1, Sat Unsat Mono Poly n

n-7a n-9 n-6 n-6 n-9 n-6 n-6 n-6 n-9 n-9 n-3 n-9

3.4. The percent composition of major fatty acids in the cellular phospholipids of epidermal and oral mucosal tissue, and plucked hair follicles

Percent total lipids In vivof

P0 (14 days)

P1–P3

2.1 9 0.5 0.0 9 0.0 44.1 9 2.2 4.7 9 0.8 16.5 90.4 21.7 9 2.2 8.3 9 1.0 0.0 9 0.0 0.1 9 0.0 0.0 9 0.0 0.1 9 0.1 0.0 9 0.0 0.2 9 0.1 0.2 9 0.1 0.8 9 0.4 0.0 9 0.0 0.0 9 0.0 1.0 9 0.5 0.5 9 0.3 64.5 93.2

5.99 0.5b 0.0 90.0b 31.29 2.5b 2.79 0.5b 19.99 0.6b 21.1 9 1.2b 4.6 90.5b 0.09 0.0b 0.69 0.3b 0.09 0.0b 0.49 0.2b 2.8 9 1.1b 4.7 90.4c 0.09 0.0b 1.69 0.6b 1.0 90.4b 2.19 0.4b 0.89 0.2b 0.69 0.2b 60.09 2.9b

4.09 0.2b 0.1 9 0.0b 23.090.4c 10.190.2c 14.7 90.2b 35.390.5c 2.19 0.3e 0.0 9 0.0b 0.89 0.3b 0.29 0.0b 1.6 9 0.1d 1.29 0.1c 2.69 0.4e 0.0 9 0.0b 0.39 0.0b 0.19 0.0b 0.9 9 0.1c 1.79 0.2b 1.6 9 0.2b 44.590.6e

35.6 93.2 26.9 92.5 8.6 9 1.1 4

40.0 92.9b 25.491.8b 12.591.5b 4

55.59 0.6e 47.490.8c 7.5 9 0.7b 6

a

The (n-) nomenclature indicates the double bond closest to methyl end. b Not significant. c 0.05\P\0.01. d 0.01\P\0.001. e P $0.05 f Plucked hairs, dissected lower portion

when compared to other intact tissue samples (Tables 1 and 2), and decreased to 23.090.4 in P1 – 3 follicle derived keratinocytes. The amount of 18:2-fatty acid in in vivo plucked hair samples is also significantly lower than that seen in epidermal and mucosal tissue. The amount of 20:4-fatty acid measured in the plucked hair in vivo samples is extremely low (0.290.1). The 20:4-fatty acid value increased in P0 cells (4.790.4), and then assumed values seen in the in vitro EFA-deficient state (compare 2.6 90.4 in Table 3to 3.190.1 in Table 1and 2.99 0 in Table 2).

The percent composition of major fatty acids in the phospholipid extracted from the three tissue types of cells after trypsin isolation is presented in Fig. 2. The basal cell pellet values of the epidermis were compared to that of the other cell pellets, and to the hair follicle fatty acid values using a two-tailed Student’s t-test. The percent of 16:0fatty acid in the hair follicles was greater than that measured in the epidermis, whereas there was no significance between the 16:0 values assayed in the epidermal cell and oral mucosal cell pellets (Fig. 2a). The three tissue types did not differ in the amount of monounsaturated fatty acids (Fig. 2b). The epidermal cell pellets and the oral mucosal cell pellets showed a significant difference in only one of the PUFAs. The percent of 18:2-fatty acid was greater in the epidermal suprabasal cell pellet when compared to the basal cell pellet from this tissue. The plucked hair follicle sample had significantly less 18:2- and 20:4-polyunsaturated fatty acids than the epidermal tissue samples (Fig. 2c). Interestingly, the suprabasal cell pellet contained a significantly larger amount of 18:2-fatty acid than the suprabasal cell pellet obtained from oral mucosa. The three types of cultured cells became EFAdeficient in culture and showed no measurable variation in total fatty acid content. There was no significant difference in any fatty acid value among three types of cultured cells (Tables 1–3, P-values not shown).

4. Discussion Several investigators have analyzed the fatty acid composition of membrane phospholipids in human skin epidermis [15,30] and oral mucosa [31,32]. However, a comparison of these lipids between the two types of the stratified squamous epithelia has not been reported. Also, there is no report describing the membrane phospholipid composition of hair follicle cells, although the

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Fig. 2. Percent composition of major fatty acids in membrane phospholipid of three types of squamous stratified epithelia. The fatty acids are extracted, TLC separated, methylated, and prepared for GC analysis as described in Section 2. Protein analysis of each sample yielded a measure of quantity that was used to estimate the sample size for the GC analysis. The data is expressed as percentage of a specific fatty acid/total fatty acid in the samples. Thus, the data is expressed as a value independent of sample size and of the variation in protein:cell ratio found among the various epithelial samples that where studied. (a) Saturated fatty acids; (b) monounsaturated fatty acids; (c) polyunsaturated fatty acids. The values are compared with one another using a two-tailed Student’s t-test: * indicates 0.05 \P \ 0.01; ** indicates 0.01 \P \0.001; no asterisk means no significance. Error bar means S.E.M.

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fatty acids in these cells should be similar to that of keratinocytes since follicle cells are thought to be the progenitor of whole skin after extensive injury [19–21]. Additionally, while the fatty acid level in cultured epidermal keratinocytes is known [15,33], these lipids have not been studied in cultured oral mucosal cells or in hair follicle cells grown in vitro. Our present studies show that as these three cell types become established in primary culture no significant difference in the percent total fatty acids was seen. GC analysis of the purified phospholipid fraction showed that these three cell types had the same fatty acid pattern, defining the EFA-deficient state [15]. Thus, the serumand fatty acid-free medium ‘imposed’ the same EFA-deficient state in all three cell types, as they grew optimally in culture. These observations suggest that stratified squamous epithelium cells grown in vitro use whatever fatty acids are available to them to synthesize phospholipids and cell membrane structures. However, other factors besides the nutritional availability of fatty acids seem to influence the fatty acid composition of in situ epithelium cells. Our analysis of the intact epithelial tissues and of cells directly removed from these tissues showed that the fatty acids in the cellular phospholipids varied, depending on the type of epithelium that was studied. Our analysis of the suprabasal and basal cell components isolated from skin epidermis found a difference in the amount of 18:2-fatty acid. The Suprabasal cells contained 35% greater amount of 18:2-fatty acid than the basal cell pellet (Table 1). This suggests that epidermal suprabasal cells accumulate 18:2-fatty acid into membrane phospholipids as a part of the differentiation program. On the other hand, there was little difference among oral mucosa suprabasal, and basal cell pellet fatty acids, and the whole tissue cells in our study (Table 2). However, the basal cell pellet was seen to contain  30% more 18:2-fatty acid than the oral mucosal suprabasal cell layer. This supports the idea that accumulation of 18:2-fatty acid is an important component of differentiation since parakeratinization has less complex differentiation process than that of the epidermis.

Our analysis of plucked hair follicles showed that there were two major and statistically significant differences in phospholipid total fatty acid composition, unique to this tissue. One was a large amount of 16:0-fatty acid, approximately twice the value seen in the other tissues (Tables 1–3, and Fig. 2a). The other was small amounts of PUFAs, less than 1/3 the values seen in skin epidermis and oral mucosal tissues (Tables 1–3 and Fig. 2c). Thus, the fatty acid composition of the hair follicle showed a partial EFA-deficient state, which was a pattern midway between that of the cultured cells and the in vivo epidermis. Although it is unclear why the amount of 16:0fatty acid is larger in the plucked hair samples, it is known that palmitic acid (16:0) is the major fatty acid which convalently binds lipids into the hair shaft, with 18-methyleicosanoic acid also accomplishing this function [28,34]. Thus, the large amount of 16:0 in the plucked hair cells may be a response to this special requirement. As the cells derived from the hair follicle grow and are passaged into P1–3, the percent of 16:0-fatty acid gradually decreases. This suggests that an excess of 16:0-fatty acid is not necessary for the hair follicle cells to differentiate into the epidermal keratinocyte-like cells that are seen in culture. Both major PUFAs were significantly decreased in the plucked hair samples. Linoleic acid (18:2fatty acid) was : 1/3 the value seen in the epidermal tissue samples and : 1/2 the value seen in the cell samples derived from oral mucosa (Tables 1–3). Arachidonic acid (20:4-fatty acid) was present at just the level of detection in the plucked hair samples (Table 3). The change in 20:4-fatty acid values seen from the plucked hair samples from P0 to P1–3 cells suggests that the enzyme involved in the conversion of 18:2- to 20:4-fatty acids is expressed in P0 cells derived from hair follicle cells. The fatty acid values of the P1–P3 cells show an EFA-deficient state identical to that seen in both the cultured epidermal and mucosal keratinocytes. The existence of this enzyme activity in keratinocytes, specifically a D desaturase activity, has been recently reported by us [35]. It has been previously reported that the hair shaft does not contain 20:4-fatty acid [28,34]. These investigators concluded that 20:4-fatty acid

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did not have an important role in forming hair. It may be suggested that since the hair follicle cells can metabolize 20:4 into 15-hydroxy5,8,11,13,eicosatetraenoic acid (15HETE), this pathway may be responsible for the low levels of 20:4-fatty acid in plucked hair although basal epidermis also has this metabolic capability [23]. Whatever the mechanism or functional cause, the plucked hair samples demonstrated a partial EFA-deficient fatty acid pattern in vivo. Because these cells are thought to be the progenitors of the epidermis in wound healing scenarios, they can be classified as putative stem cells. We hypothesize that the altered fatty acid composition reported here for plucked hair is one characteristic of the epidermal stem cell state, and is an important component of the environment necessary for the maintenance of the hair structure. We believe that our data is informative in obtaining an understanding the role of membrane fatty acids in the function of stratified squamous epithelium. It is hoped that since hair is composed mainly of keratins and various types of lipids, our data describing the hair follicles in vivo would be useful in developing future organ culture systems [36] and in developing technology to better preserve the entire hair structure [37].

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